In vitro CPE assay in Vero 76 cells

The antiviral activities of test compounds were evaluated in Vero 76 cells. Eight half-log dilutions were tested and the effective antiviral concentration determined by regression analysis. Briefly, compounds were diluted on nearly confluent monolayers of Vero 76 cells and infected with a multiplicity of infection (MOI) of 0.001 of SARS-CoV-2. Five microwells were used per dilution with three wells used for infected cultures and two wells used for toxicity. Plates were incubated at 37°C with 5% CO₂ for 5 days. Plates were stained with 0.011% neutral red for two hours and dye eluted with 50% Sorensen’s citrate buffer/50% ethanol for 30 minutes. The dye content in each well was converted to a percentage of dye in untreated control wells and normalized to virus control. 50% cytotoxic and 50% effective concentrations were determined by regression analysis using an excel spreadsheet.